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Pharmacological Research 56 (2007) 318–328

Synthesis, in vitro activity and in vivo toxicity of the new2,3-dinitrobutadiene derivative

(1E,3E)-1,4-bis(2-naphthyl)-2,3-dinitro-1,3-butadiene

Maurizio Viale a,∗, Giovanni Petrillo b, Cinzia Aiello a, Carla Fenoglio c,Cinzia Cordazzo a, Maria A. Mariggio d, Amalia Cassano d, Claudia Prevosto e,

Emanuela Ognio f, Massimo Maccagno b, Lara Bianchi b, Rita Vaccarone c,Egon Rizzato g, Domenico Spinelli g

a Istituto Nazionale per la Ricerca sul Cancro, S.C. Terapia Immunologica, L.go R. Benzi 10, 16132 Genova, Italyb Dipartimento di Chimica e Chimica Industriale, Universita di Genova, Via Dodecaneso 31, 16146 Genova, Italy

c Dipartimento di Biologia Animale, Universita di Pavia, P.za Botta 10, 27100 Pavia, Italyd Dipartimento di Scienze Biomediche e Oncologia Umana, Universita di Bari, Osp. Policlinico, P.za G. Cesare 11, 70124 Bari, Italy

e Istituto Nazionale per la Ricerca sul Cancro, S.C. Oncologia Sperimentale D, L.go R. Benzi 10, 16132 Genova, Italyf Istituto Nazionale per la Ricerca sul Cancro, S.S. Sperimentazioni su Modelli Animali, L.go R. Benzi 10, 16132 Genova, Italy

g Dipartimento di Chimica Organica “A. Mangini”, Universita di Bologna, Via San Giacomo 11, 40126 Bologna, Italy

Accepted 31 July 2007

bstract

Our interesting results on the antiproliferative (in vitro) and antitumour (in vivo) activities of (1E,3E)-1,4-bis(1-naphthyl)-2,3-dinitro-1,3-utadiene (1-Naph-DNB) have more recently induced us to design and synthesize some new 1,4-diaryl-2,3-dinitro-1,3-butadienes characterizedy a common arylnitrobutadiene array but with different geometric and/or functional properties. This task was undertaken with the aim to obtainew compounds with an enhanced antiproliferative activity and, possibly, a different specificity with respect to the original (lead) compound.1E,3E)-1,4-Bis(2-naphthyl)-2,3-dinitro-1,3-butadiene (2-Naph-DNB) is one of the molecules so obtained, a structural isomer of 1-Naph-DNBrovided with a different spatial arrangement. When analyzed in vitro for its inhibition of cell proliferation 2-Naph-DNB showed a remarkablectivity in the range of micromolar concentrations, with significant differences, with respect to 1-Naph-DNB, against some cell lines. Furthermore,t was able to significantly trigger apoptosis, to up-regulate p53, to block cells in the G2/M phase of the cell cycle and, finally, to slightly bind toNA forming interstrand cross-links (ISCL).2-Naph-DNB was then analyzed for its toxic activity in vivo in CD1 mice. This allowed the determination of toxicity parameters such as the

ethal doses (LD) and the maximal tolerated dose (MTD) together with the definition of the spectrum of tissue alterations due to its administration.v.

Altogether our data suggest that the idea of modifying the geometry of the lead compound 1-Naph-DNB deserves further investigation aimed atynthesizing new molecules with similar chemical functionalities but with different spatial requirements, hopefully characterized by still enhancedctivities in terms of inhibition of cell proliferation and apoptosis.

2007 Elsevier Ltd. All rights reserved.

icity

eywords: Naphthylnitrobutadienes; Antiproliferative activity; Apoptosis; Tox

∗ Corresponding author. Tel.: +39 010 5737320.E-mail address: maurizio.viale@istge.it (M. Viale).

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043-6618/$ – see front matter © 2007 Elsevier Ltd. All rights reserved.oi:10.1016/j.phrs.2007.07.009

. Introduction

In spite of an intensive and capillary information about pri-

ary prevention, the increased risk of exposure to carcinogens

ue to the explosive progress of life conditions in developedountries and the consequent global environmental modifica-ions have constantly and significantly increased the incidence

M. Viale et al. / Pharmacological R

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Fig. 1. Molecular structures of 1-Naph-DNB and 2-Naph-DNB.

f cancer (which is the main cause of mortality after cardio-ascular diseases) in the last few years [1–4]. Many tumouristotypes, such as those of stomach, brain, pancreas, colorec-um, and lung, have still a bad prognosis associated with annacceptable high mortality. This is also because the range ofhemotherapeutic weapons nowadays available does not yetover all possible intrinsic or acquired forms of resistance torugs [5–7].

Obviously, in these conditions the discover of new moleculartructures endowed with significant antiproliferative activitiesnd specificity against new tumour histotypes still remains anssential goal of pharmacological research [8,9].

In previous papers [10–13] we have reported on the antipro-iferative activity in vitro and on the antitumour activity in vivof 1-Naph-DNB (Fig. 1). This molecule showed a pharmacolog-cally significant activity in vitro in terms of inhibition of cellroliferation, cytotoxicity and induction of apoptosis, togetherith a particular selectivity for cell lines derived from otherwisenresponsive gastrointestinal tumours. This last result was inart confirmed in vivo [13].

The enlightened ability of 1-Naph-DNB to form interstrandross-links with the DNA of tumour cells as the most likelyechanism of apoptosis [10] has suggested the opportunity to

onsider its molecular structure as a prototype for the designf novel molecules in search for new and/or enhanced pharma-ological activities in terms of intensity and specificity. Withinhis research line, we have now synthesized the isomeric 2-Naph-NB (Fig. 1), characterized by the same chemical functionalities

s 1-Naph-DNB [i.e. by the presence of a (1E,3E)-2,3-dinitro-,3-butadiene moiety flanked by two polycyclic aromatic rings]ut by a molecular arrangement of the two naphthyls whichould allow a different adaptability to the DNA interstrandavity.

Herein we analyze the antiproliferative activity of 2-Naph-NB together with its ability to trigger apoptosis, to up-regulate53 protein, to cause modifications of the cell cycle and toind to DNA forming ISCL. Moreover, data about the lethal

meam

Scheme 1

esearch 56 (2007) 318–328 319

nd toxic effects in vivo of 2-Naph-DNB have also beenbtained.

. Materials and methods

.1. Chemistry

Likewise 1-Naph-DNB, 2-Naph-DNB is a stable, fullyharacterized diaryldinitrobutadiene deriving from the initialing-opening of 3,4-dinitrothiophene [14–17]. The syn-hesis of 2-Naph-DNB has been achieved via arylationf 1,4-bis(diethylamino)-2,3-dinitro-1,3-butadiene with 2-aphthylmagnesium bromide (see Scheme 1) as describedereinafter.

A solution of (1E,3E)-1,4-bis(diethylamino)-2,3-dinitro-,3-butadiene (1 mmol, 300 mg; synthesized from 3,4-initrothiophene as previously reported in Ref. [14]) inetrahydrofuran (THF, 10 ml) was cooled to 0 ◦C under mag-etic stirring. The calculated amount of a ca. 0.5 M solution of-naphthylmagnesium bromide (2.2 mmol) in THF was slowlydded by syringe and the reaction mixture left under stirringt the same temperature until disappearance of the substrateca. 1 h), the progress of the reaction being monitored byLC. Quenching of the reaction was performed by pouring

he final solution, with vigorous shaking, into a mixture ofichloromethane/ice water containing 2.2 mmol of HCl. Afterxtraction with dichloromethane, the organic phase was washedith water, dried with Na2SO4 and rotoevaporated to dryness.he product (98% yield) is obtained in quite pure form byhromatography of the residue on a silica gel (63–200 mesh) col-mn eluted with a gradient of dichloromethane and petroleumther. 2-Naph-DNB is a yellow solid, mp 179–180 ◦C (lightetroleum/toluene); 1H NMR (CDCl3) δ 7.41–7.56 (6H, m),.68–7.75 (6H, m), 7.90 (2H, s), 8.71 (2H, s); 13C NMR (CDCl3)124.5, 127.1, 127.2, 127.8, 128.9, 129.0, 129.4, 132.8, 133.8,34.7, 140.1, 141.6. ESI (Electrospray Ionization)-MS: m/z18.9 (M + Na+), 815.1 (2M + Na+).

1H NMR and 13C NMR spectra were recorded on a Varianercury 300 spectrometer at 300 MHz and 60 MHz, respec-

ively (TMS as internal reference). The ESI-MS spectrumas acquired on a ZMD Micromass single quadrupole mass

pectrometer operating at 4000 m/z. The melting point was deter-

ined with a Buchi 535 apparatus and is uncorrected. Petroleum

ther and light petroleum refer to the fractions with bp 40–60 ◦Cnd 80–110 ◦C, respectively. THF was purified by standardethods and distilled over potassium benzophenone ketyl before

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20 M. Viale et al. / Pharmacolog

se. All other commercially available reagents were used aseceived.

.2. In vitro

.2.1. Growth inhibition assayNine human and murine cell lines (human: A2780, (ovary,

arcinoma) [18], A549 (lung, carcinoma) [19], H1299 (lung,arcinoma, kindly provided by Dr. K. Okaichi) [20], HGC-7 (stomach, carcinoma) [21], HCT-8 (colon, adenocarcinoma)22], Jurkat (T cell leukaemia) [23], MDA-MB-231 (breast, ade-ocarcinoma) [24], PA-1 (ovary, teratocarcinoma) [25]; murine:388 (leukaemia) [26]) were plated into 96-well microtiterlates at different concentrations/well (range: 1500–5000/well)or about 8 h. 2-Naph-DNB was added in duplicate at theppropriate concentrations for a minimum of five concentra-ions (two to three-fold serial dilutions). A final volume of00 �l was reached in each well. After 3-day culture 50 �l of-(2,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bro-ide (MTT, Sigma, St. Louis, MO, USA) solution [2 mg/ml in

hosphate buffered saline (PBS)] was added to the wells andncubated for 4 h at 37 ◦C. Microplates were then centrifuged at75 g for 5 min and the culture medium carefully aspirated andeplaced with 100 �l of 100% dimethylsulfoxide. Complete andomogeneous solubilization of formazan crystals was achievedfter 20 min of incubation and shaking of well contents. Thebsorbance was measured on a 400 ATC microculture plateeader (SLT Labinstruments, Austria) at 540 nm [27]. IC50’sere calculated by the analysis of single-dose response curves,

ach final value being the mean of 4–11 independent experi-ents.

.2.2. Trypan blue (TB) dye exclusion assay andisualization of apoptotic cells/nuclei by′-6-diamidino-2-phenylindole (DAPI) staining

Cell lines were plated at different cell concentrations/wellrange: 10,000–30,000/well) into 24-well microtiter plates forbout 8 h, then 2-Naph-DNB was administered at its specificC50 and IC75, as calculated with the MTT assay. After 72 hells were harvested, washed twice with cold PBS and splitted inwo halves. One half of cells was concentrated to 100 �l normalaline and stained with 5 �l of TB (0.4% in 0.81% sodium chlo-ide and 0.06% potassium phosphate dibasic). The percentagef stained dead cells was evaluated at the microscope.

The other half was fixed with 100 �l of 70% ethanol inBS and maintained at 4 ◦C. For the examination at the micro-cope, 5 �l of a solution of 10 �g/ml DAPI in water was addednd immediately after the percentage of apoptotic segmenteduclei/cells was evaluated.

.2.3. Western blot analysis of p53Human A2780, A549, Jurkat, MDA-MD-231, PA-1 and

GC-27 cells were treated with their specific IC50s of 2-

aph-DNB, as previously evaluated by the MTT assay. After4 and 48 h culture control and treated cells were harvested,ashed twice with cold PBS and treated with a lysis buffer

1% Triton X-100, 0.15 M NaCl and 10 mM Tris (pH 7.4)]

t93(

esearch 56 (2007) 318–328

ontaining protease inhibitors (50 mg/ml phenylmethylsulpho-yl fluoride and 2 mg/ml aprotinin) for 30 min at 4 ◦C. Therotein concentration was determined by the Bradford methodSigma Chemical Co., St. Louis, MO, USA). Equal amountsf total protein (30–60 �g) were separated on a 12% poly-crylamide gel (SDS-PAGE). Proteins were transferred onto aitrocellulose membrane (Hybond C-Extra, Amersham Italiarl, Milan, Italy) and protein loading checked by Ponceau Staining. Nonspecific binding was blocked overnight at 4 ◦C with% bovine albumin (Sigma) in Tris–buffered saline–Tween 200.15 M NaCl, 10 mM Tris (pH 8.0), 0.05% Tween 20]. Blotsere probed with anti-p53 DO-1 (1:2000, Santa Cruz, CA,SA) monoclonal antibodies. After incubation with horseradisheroxidase-conjugated antimouse IgG, bands were visualizedy chemiluminescent detection (ECLTM Western blotting anal-sis system, Amersham Italia Srl) following the supplier’secommended procedures. Prestained molecular weight mark-rs (New England Biolabs, Beverly, MA, USA) were used aseference.

Alternatively, A549 cells were incubated for 72 h with differ-nt concentrations of 2-Naph-DNB. Cells were then treated and53 analyzed as described before.

Densitometric normalization was performed by using thexpression signal obtained by an anti-�/�-tubulin monoclonalntibody (TU-10,1:5000, Santa Cruz, CA, USA).

.2.4. Analysis of the cell cycleA549, Jurkat and HGC-27 cells were also analysed for the

ffect of 2-Naph-DNB on cell cycle phases. Cells (1–2 × 106)ere treated for 48 h with three different concentrations of ourolecule, harvested, washed twice with cold PBS and fixed in

0% ethyl alcohol at −20 ◦C overnight. After fixation, cells wereentrifuged, washed with PBS, and incubated at room temper-ture for 20 min with propidium iodide (PI) staining solution50 �g/ml PI, 0.05% Triton X-100, 20 mg/ml RNase A in PBS).ells were analyzed by flow cytometric analysis of DNA contentsing FACSort flow cytometer (BD Bioscences, Mountain View,A). The percentages of cell cycle distribution were calculatedy ModFit LT computer program.

.2.5. Analysis of interstrand cross-link (ISCL)The % ISCL was evaluated using, as target, pure DNA

xtracted from P388 cells by the salting out technique [28]260/280 = 1.84) and dissolved in distilled water. Two differentoncentrations (1.0 and 0.1 �M) of 2-Naph-DNB were incu-ated with DNA (final concentration: 485 �g/ml) for 1 h at7 ◦C, then DNA was precipitated with sodium acetate andthanol and resuspended in Tris–buffered saline–Tween 20. Aolution (1.5 ml) of ethidium bromide (10 �g/ml in 0.4 mMDTA, 20 mM K2HPO4, pH 11.8) was added to 0.2 ml (10 �g)liquots of DNA extracted from control and treated cells.he fluorescence was measured before and after heating at5 ◦C for 8 min (Fluoroskan Ascent, Thermolab Systems; exci-

ation wavelength, 530 nm; emission wavelength, 590 nm) in6 microtiter plates, while the final volume in the wells was00 �l. The % ISCL was determined according to the formulaft − fn)/(1 − fn) × 100, where ft and fn were the fluorescence

ical Research 56 (2007) 318–328 321

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Table 1In vitro determination of inhibition of cell proliferation (MTT) by 2-Naph-DNB

Cell line Histotype/origin 2-Naph-DNB

IC50 IC75

P388 Leukemia/mouse 1.7 ± 0.4a 3.4 ± 0.1A2780 Ovary/human 3.9 ± 1.0 5.9 ± 1.5PA-1 Ovary/human 2.9 ± 0.8 4.6 ± 0.9Jurkat Leukemia/human 5.1 ± 1.2 11.7 ± 2.1HGC-27 Stomach/human 4.7 ± 0.8 7.2 ± 1.6MDA-MB-231 Breast/human 5.9 ± 1.6 12.3 ± 2.9HCT-8 Colon/human 13.9 ± 1.6 19.8 ± 4.4A549 Lung/human 13.4 ± 3.4 22.5 ± 6.7H

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Finally, we analyzed the induction of apoptosis by the obser-vation of nuclear segmentation after DAPI staining. Althoughonly Jurkat cells showed a high sensitivity when exposed to the

M. Viale et al. / Pharmacolog

fter denaturation divided by the fluorescence before denatura-ion of treated (ft) and control (fn) samples.

.3. In vivo

.3.1. AnimalsFemale CD1 mice (20–25 g, Harlan Italy, S. Pietro al

atisone, Italy) were allowed a 7-day period before use.tandard food and water ad libitum were available at all

ime. All experiments were performed in accordance with theuidelines of the Federation of European Laboratory Animalcience Associations and approved by the Institutional Reviewoard for animal studies.

.3.2. Toxicological testingThe determination of lethal and maximal tolerated doses was

erformed in normal female CD1 mice by daily observationf toxicity sign and survival. Groups of 8 + 2 satellite animalsere treated i.v. with a single injection of 2.5, 5, 7.5, 10 and5 mg kg−1 of 2-Naph-DNB. LD10, LD50 and LD90 parametersere calculated using deaths up to 14 days after administration.y the same experiments we also evaluated the MTD, as theaximal administered dose of Naph-DNB without significant

oxic effects.2-Naph-DNB was diluted in DMSO and given in a volume

ot exceeding 1 �l g−1 of body weight in order to avoid toxicffects of the solvent. Body weight was recorded every 2 days.

Satellite animals were sacrificed with CO2 on day 4 or whenying. Immediately after autopsy fresh tissues obtained fromrgan explants were fixed and processed for histological analy-is.

.3.3. Light microscopyFor light-microscopy studies, fragments of liver, kidney,

pleen, lung and heart were fixed for 24 h in 2% paraformalde-yde in 0.1 M PBS, pH 7.4, processed routinely, embedded inaraplast wax, and sectioned at 6 �m.

Sample sections of all the tissues were stained with hema-oxylin and eosin (H&E). Moreover, the Perls’ Prussian Blueeaction was performed on samples from liver and spleen, toetect ferric pigments content. Liver sections underwent PAStaining (with and without �-amylase digestion) to detect glyco-en; PAS reaction was performed also on kidney sections. Allhe samples were evaluated qualitatively.

.4. Statistical analysis

The Mann–Whitney and Friedman ANOVA & Kendall’soncordance tests for not parametric data and the unpairedtudent’s t test were used for statistical analysis of data.

. Results

.1. In vitro

.1.1. Antiproliferative activity and induction of apoptosisThe IC50 values obtained from the concentration–response

urves designed after the treatment of the different cell lines

FcI

1299 Lung/human 3.6 ± 0.8 10.1 ± 2.2

a Data express the mean ± S.D. of 4–11 experiments.

ith 2-Naph-DNB showed that this molecule is characterizedy a good antiproliferative activity, with IC50s and IC75s rang-ng in the 1.7 ± 0.4–13.4 ± 3.4 �M range of concentrationsMedian IC50 = 4.7 �M) and 3.4 ± 0.1–22.5 ± 6.7 �M (MedianC75 = 10.1 �M), respectively (Table 1). The cell lines most sen-itive to the exposure to 2-Naph-DNB were P388, A2780 andA-1. A good antiproliferative activity was also recorded againsthe p53-null human H1299 lung carcinoma cell line (Table 1).

A similar result was obtained considering the cytotoxic activ-ty, as determined by the trypan blue dye exclusion assay,nd exposing cells to their specific IC50 (Median % TB+

ells = 20%) and IC75 (Median % TB+ cells = 40%), as deter-ined by the MTT assay. Also in this case P388, A2780, and

urkat proved to be most sensitive to the cytotoxic activity of-Naph-DNB. Interestingly, also H1299 showed a discrete sen-itivity to the cytotoxic activity of our nitrobutadiene compoundFig. 2).

ig. 2. Histograms represent the percentage of cells stained with the TB (�, deadells) or DAPI (�, apoptotic cells) dyes after 72 h exposure to the correspondingC50s of 2-Naph-DNB.

322 M. Viale et al. / Pharmacological R

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ig. 3. Histograms represent the percentage of cells stained with the TB (�, deadells) or DAPI (�, apoptotic cells) dyes after 72 h exposure to the correspondingC75s of 2-Naph-DNB.

C50 of 2-Naph-DNB (as evaluated by the MTT assay), whene applied the IC75 the percentage of apoptotic cells increased

ignificantly, particularly in P388, PA-1, Jurkat, and A549 cellsrange: 23–90%) (Fig. 3). As expected, no apoptotic cells wereecorded when p53-null H1299 cells were treated with bothoncentrations.

.1.2. Western blot analysis

Analysis by western blot of p53 oncoprotein was performed

n A549, A2780, PA-1, MDA-MB-231 and Jurkat cells. In allell lines 2-Naph-DNB was able to up-regulate p53 with relativeaximal increases of p53 concentration ranging from +5% to

tieN

able 2ercentage of A549, Jurkat, and HGC-27 cells in the different cell-cycle phases after

ell line Cell-cycle phase Concentration of 2-Nap

0

549G0/G1 40.7 ± 6.5a

S 37.1 ± 0.8G2/M 22.2 ± 6.5

ell line Cell-cycle phase Concentration of 2-Nap

0

JurkatG0/G1 41.6 ± 4.8

S 36.1 ± 4.7G2/M 22.3 ± 2.1

ell line Cell-cycle phase Concentration of 2-Nap

0

GC-27G0/G1 37.1 ± 0.9S 38.1 ± 0.8G2/M 24.8 ± 0.5

a Mean ± S.D. of 3/4 data.b p < 0.05, as determined by the Mann–Whitney-test for not parametric data.

esearch 56 (2007) 318–328

245%. In terms of protein increase, cells with wild type p53A2780, A549 and PA-1) seem to have an oncoprotein upregu-ation higher than that observed in MDA-MB-231 provided of a

utated form of p53.Maximal protein increase (Fig. 4A) was reached at different

imes for different cell lines and, in spite of the limited number ofime points used for this determination, our data reveal differentinetics for the upregulation of p53. Moreover, upregulation waslearly concentration-dependent, as demonstrated in A549 cellsfter 72 h exposure (Fig. 4B).

.1.3. Cell cycleA549, Jurkat and HGC-27 cells were also analyzed for the

odifications of cell-cycle phase after exposure to increas-ng concentrations of 2-Naph-DNB. As shown in Table 2, ourata display that 2-Naph-DNB was able to significantly causepartial block of cells in the G2/M phase of the cell cycle,ith a concomitant decrease of cells in the S phase. While

n HGC-27 cells this effect showed a clear trend toward aorrelation with the applied concentrations of 2-Naph-DNBp < 0.07), in A549 and Jurkat cells it becomes significant ver-us control cells only at the higher concentrations (p < 0.05)Table 2).

.1.4. Interstrand cross-links2-Naph-DNB was tested for its ability to form ISCL. In a

ree-cell system using pure DNA isolated from P388 cells asarget, our compound was able to form ISCL, although the %SCL recorded was lower than expected (Fig. 5) if compared

o the results obtained with 1-Naph-DNB [10]. Moreover, its noteworthy that when the % ISCL was evaluated on DNAxtracted from A549, A2780 and Jurkat cells treated with 2-aph-DNB for 6 h at their IC50 (as calculated by the MTT assay)

treatment with different concentrations of 2-Naph-DNB

h-DNB (�M)

6.25 25 100

37.7 ± 3.9 50.6 ± 5.8 34.5 ± 0.3

38.7 ± 1.2 29.2 ± 5.1 20.3 ± 1.5b

23.6 ± 4.6 20.2 ± 0.4 45.3 ± 1.8b

h-DNB (�M)

0.94 3.75 15

40.6 ± 5.5 35.3 ± 1.5 46.3 ± 3.4

37.1 ± 6.4 41.3 ± 2.4 23.1 ± 4.7b

22.3 ± 2.3 23.4 ± 2.8 30.6 ± 6.6b

h-DNB (�M)

1.88 7.5 30

33.8 ± 4.3 36.2 ± 1.8 46.8 ± 1.235.2 ± 5.5 30.6 ± 2.8b 23.8 ± 2.9b

31.2 ± 1.6b 33.2 ± 1.7b 29.4 ± 2.5b

M. Viale et al. / Pharmacological R

Fig. 4. (A) Western blot analysis of p53 after exposure of different cell lines to thecorresponding IC50s of 2-Naph-DNB, calculated by the MTT assay. Prestainedmolecular markers were always included as references. Control was harvestedafter 48 h. (B) Representative Western blot analysis of p53 after exposure ofA549 cells to increasing concentrations of 2-Naph-DNB.

Fig. 5. Histograms represent the % ISCL obtained exposing pure DNA extractedfrom murine P388 cells to different concentrations of 2-Naph-DNB for 1 h.Histograms represent the mean ± S.D. of three data.

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esearch 56 (2007) 318–328 323

he % ISCL recorded was again lower than that observed forhe parent compound 1-Naph-DNB (in general < 1%, data nothown).

.2. In vivo

.2.1. Toxicity studiesBased on the percentage of died CD1 mice treated with single

dministration of 2-Naph-DNB i.v., we determined the LD andTD shown in Table 3. By the same experiments we also cal-

ulated both the MTD and the percent variation of body weightTable 3).

Extravasation of 2-Naph-DNB caused a reversible caudallcer and, as in the case of 1-Naph-DNB [12], it was not com-letely healed 14 days after administration (not shown).

.2.2. Morphology and histochemistryFrom a morphological point of view all the organs of the

ontrol animals showed a normal structural aspect (data nothown).

Histochemical analysis of control liver revealed typicallycogen zonation within the parenchyma submitted to PASeaction. In particular, cells surrounding pericentral vein wereore intensely stained compared to periportal cells. In the same

ections, very light Perls’ reactivity was generally observed inhe hepatocytes and endothelial cells.

The liver parenchyma of samples treated with low dosesf 2-Naph-DNB (2.5 and 5 mg kg−1) was structurally similaro controls (Fig. 6A). PAS positivity was still mainly concen-rated in pericentral hepatocytes, though a slight decrease inntensity was observed after treatment with 5 mg kg−1 of 2-aph-DNB (Fig. 6B). After Perls’ method a light cytoplasmic

taining occurred in hepatocytes. More intense reactivity waseen in sinusoidal cells (Fig. 6C).

Severe changes were noted in parenchyma of mice treated−1

ith the highest doses of 2-Naph-DNB (7.5 and 10 mg kg ). A

rocess of hepatocytic alteration affected the parenchyma irregu-arly, mainly at 10 mg kg−1. Most cells displayed light cytoplasmnd several nuclei were apoptotic. Moreover, sinusoids and some

able 3etermination of lethal doses (LD) and maximal tolerated dose (MTD) in CD1ice treated with single doses i.v. of 2-Naph-DNB (upper table) and percenteight loss on days +2 and +4

oute of administration LD10 LD50 LD90 MTD

.v. 3.5a 6.8 11.9 2.8

ay of observation Doses of 2-Naph-DNB (mg kg−1)

2.5 5 7.5 10 20

2 −1.8 −0.1 −3.9 −11.5 –b

4 +1.7 +3.3 −2.7 –c –

a Values represent the doses expressed in mg kg−1. Animals were treated andhen observed for 14 days.

b All animals died by day +2.c All animals but two died by day +4.

324 M. Viale et al. / Pharmacological Research 56 (2007) 318–328

Fig. 6. Liver from representative treated mice with 5 mg kg−1 (A–C) and 10 mg kg−1 2-Naph-DNB (D–F); sections stained with H&E (A and D), PAS reaction(B and E) and Perls’ technique (C and F). At low dose the hepatocytes surrounding both portal tract (P) and central vein (CV) show no pathological changes;m , Perl1 s ando ue iro

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oderate/intense perivenous PAS staining is noted in most hepatocytes. In (C)0 mg kg−1 2-Naph-DNB show altered hepatocytes, sinusoids filled with red cellbserved in the parenchyma. In (F), note large hepatocytes that show intense bl

essels appeared greatly congested with erythrocytes, and a mildnfiltration of leukocytes was sometimes observed in portal areasFig. 6D). PAS staining displayed an irregular distribution oflycogen content around the central vein, caused by the pres-nce of hepatocytes with high staining intensity among mostepatocytes with considerably lower positivity (Fig. 6E). Ineneral, light to moderate Pearls’ reactivity was observed inhe parenchyma, except for occasional large hepatocytes thatontained abundant blue deposits (Fig. 6F).

The spleen parenchyma of both control animals and thosereated with low doses of 2-Naph-DNB showed distinct areas ofhite and red pulp (Fig. 7A). The red pulp was characterized byoderate to intense Perls’ reactivity, mainly in the cells of the

eticulo-endothelial system. Light positivity was also observed

long blood capillaries in the white pulp (Fig. 7B). However,tarting from the dose of 5 mg kg−1 numerous megakaryocytesere observed within the red pulp (Fig. 7C). At the highest doses

pleens exhibited partial disruption of follicular architecture and

4

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s’ staining is evident in the endothelial cells. Sections from mice treated withleucocytes infiltration in the portal (P) space (arrow). PAS staining is irregularlyn staining (arrowheads). A, B, D and E, 100×; C and F, 200×.

he red pulp displayed a remarkable congestion of erythrocytesFig. 7D). In the same samples the Perls’ staining was diminishedFig. 7E). Moreover, in the white pulp the capillaries appearedilated and contained leucocytes (Fig. 7F).

The morphology of the kidney parenchyma of samples treatedith the lowest dose of 2-Naph-DNB was similar to control

Fig. 8A,B). Starting from the dose of 5 mg kg−1 occasionalroximal tubules displayed epithelia cells with light cytoplasmnd irregular brush border. At the highest doses of 2-Naph-DNB7.5 and 10 mg kg−1) more tubules appeared altered, and mostapillaries intermingled with tubules were dilated (Fig. 8C). Inarticular, the PAS reaction evidenced irregular profiles of thepithelia constituting the proximal tubules (Fig. 8D).

. Discussion

Our recent data [10–13] both in vitro and in vivo have shownhat 1-Naph-DNB possesses a significant antitumour activity

M. Viale et al. / Pharmacological Research 56 (2007) 318–328 325

Fig. 7. Spleen from representative treated mice with 5 mg kg−1 (A and B), 7.5 (C) and 10 mg kg−1 2-Naph-DNB (E and F); sections stained with H&E (A and D),and Perls’ technique (B, C, E and F). Characteristic morphology of the spleen parenchyma is noted in samples treated with low dose; the white pulp (WP) shows adefinite structural organization and is surrounded by the red pulp (RP) (A). Very intense Perls’ staining is observed in the red pulp (B, thick arrow). After the treatmentw pulp (( ularw

mv

cro1atteavi

d

toNdsuIiaeMc

ith 7.5 mg kg−1 2-Naph-DNB note the presence of megakaryocytes in the redD). While the red pulp shows a reduced Perls’ reactivity, it is to note the irreghite pulp (F, arrowheads). A, B, D and E, 100×; C and F, 600×.

ainly based on its binding to DNA and on the following acti-ation of the apoptotic machinery.

In the perspective of using 1-Naph-DNB as a leadompound, purposely devised functional modifications haveevealed [unpublished data] that the pharmacological activityf such molecules essentially rests on the 1-naphthyl-2-nitro-,3-butadiene array. In this line, 2-Naph-DNB has been deviseds a purely structural (rather than functional) modification ofhe lead, possibly able to determine a better adaptability ofhe molecule within the tumour-cell DNA double strand, thusnforcing, in particular, a pharmacologically more significantntiproliferative activity. Such an outcome would definitely pro-

ide new anticancer compounds to be confidently subjected ton vivo experiments.

As a matter of fact, our outcomes show that 2-Naph-DNB isefinitely less capable than 1-Naph-DNB to form ISCL when

sHa1

C, arrows). With the highest dose, the RP appears congested with erythrocytesorganisation of the white pulp (E, thick arrow). Note dilated capillaries in the

ested in a cell-free system on pure DNA or when tested directlyn cells. Nonetheless, in spite of its lower ability to form ISCL, 2-aph-DNB possesses a good antiproliferative activity (althoughifferent in terms of specificity if compared to 1-Naph-DNB:ee below in the discussion) also when this activity is eval-ated in terms of cytotoxicity and/or induction of apoptosis.n particular, the in vitro behaviour of 2-Naph-DNB interest-ngly suggests that this compound is able to ensure a goodntiproliferative activity against various tumour cells of differ-nt histological origin. Fig. 9 illustrates, in terms of IC50 for theTT assay, the relative change in specificity of 2-Naph-DNB

ompared to 1-Naph-DNB: thus, while more pharmacologically

ignificant effects of the 2-naphthyl derivative were found inGC-27 and MDA-MB-231 cells, the prevalent antiprolifer-

tive activity in A2780, Jurkat and HCT-8 cells was due to-Naph-DNB.

326 M. Viale et al. / Pharmacological Research 56 (2007) 318–328

F and wA highe , 100

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FeAp

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ig. 8. Kidney from representative treated mice with 2.5 mg kg−1 (A and B)fter the low dose treatment the parenchyma shows normal morphology. With

specially proximal tubules display irregular luminal profiles (arrows). A and B

Therefore our data, while not excluding a possible interac-ion with the tumour-cell DNA, on the other hand open theossibility of a mechanism of action different from that hypoth-sized for 1-Naph-DNB. Such a result could be also supportedy our unpublished data from our laboratory about the cross-

esistance in taxol-resistant cells, made resistant in vitro byeans of the induction of a point mutation of �-tubulin able

o alter the stability of microtubules. This in turn suggests the

ig. 9. Comparison between the IC50s calculated by the MTT assay after 72 hxposure to 1-Naph-DNB (�) and 2-Naph-DNB (�). p-values: P388, p < 0.05;2780, p < 0.01; PA-1, NS; Jurkat, p < 0.01; HGC-27, p < 0.001; MDA-MB-231,< 0.01; HCT-8, p < 0.001; A549, NS.

tapbdapts2tmtaDr

a(staac

bd

ith 10 mg kg−1 (C and D) 2-Naph-DNB; sections stained with PAS reaction.dosage peritubular vessels appear dilated (arrowheads); numerous tubules, and×; B and D, 200×.

ossibility of previously unveiled mechanisms of action for-Naph-DNB.

Although some doubts may still remain about the effectiveole of apoptosis in clinical setting, the hypothesis that thishysiological pathway of cell death may be of relevance tohe response to anticancer drugs in human patients is generallyccepted [29–34]. This is the reason why one property that com-ounds with antiproliferative properties must have, in order toe considered deserving of a possible development as anticancerrugs, is the ability to trigger apoptosis. This feature has actu-lly been confirmed herein, 2-Naph-DNB being able to activate53 (in a concentration-dependent manner and with a differentiming of upregulation in different cells) and to induce nuclearegmentation, the typical nuclear morphology of apoptotic cells.-Naph-DNB seems also definitely able to up-regulate p53 bet-er in cells with a wild type form than in cells endowed with a

utated form, although the low number of cell lines involved inhis study may not allow final conclusions about this point. Thectivation of p53 could confirm the binding of 2-Naph-DNB toNA and its damage in spite of the reduced formation of ISCL

ecorded by our experiments.On the other hand, our finding that the percentage of cells in

poptosis (as evaluated by DAPI staining) was about nine times2% versus 17.5% at the IC50) lower than the percentage of cellstained by the TB dye in p53-null H1299 cells suggests thathe oncosuppressor gene p53 is critical for the activation of thepoptotic machinery by 2-Naph-DNB. Moreover, the fact thatlso at high concentration (IC75) we did not observe apoptotic

ells (Fig. 3) strengthens this conclusion.

Furthermore, the ability of our nitrobutadiene compound tolock cells in the G2/M phase of the cell cycle in a concentration-ependent manner represents a rather strong hint for an effective

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M. Viale et al. / Pharmacolog

ink between DNA (or other targets to be identified) damage, p53ctivation, block in G2/M phase of the cell cycle and apoptosis.

Altogether, the data collected seem to nicely fulfil ourxpectations, suggesting that a purely structural (rather thanunctional) modification of the 1-Naph-DNB lead, devised withhe hope to increase the link or intercalation of 2-Naph-DNBo DNA, may significantly influence the antiproliferative activ-ty of the original molecule, in particular against HGC-27 and

DA-MB-231 cells, i.e. those derived from tumour histotypesess responsive to chemotherapeutics.

Our in vivo toxicity experiments allowed both the definition ofDs and of MTD parameters useful for the following evaluation

n vivo and the analysis of tissue damage due to treatment withncreasing doses of 2-Naph-DNB. On the basis of histochemi-al analysis prominent changes have regarded liver and spleen,lthough depending on the dosage used. At the highest doses,he presence of congested vessels in both the organs suggestspossible mechanism of toxicity due to a progressive ischemic

njury. However, in the liver we cannot exclude a primary inter-ction of the molecule with the hepatocytes, as evidenced byhe altered PAS positivity, the elevated Perls’ reactivity of someepatocytes, and the presence of apoptotic cells. On the otherand, the prominent involvement of this organ in the detoxify-ng processes makes hepatocytes sensitive to drugs mainly ifdministered at high dosage.

Changes in haemopoietic/haemocatheretic engagement ofpleen, mainly observed in high-dose treated mice, are prob-bly related to changes of the vascular conditions in both livernd spleen. In particular, while the distribution of the Perls’eaction in spleen of control and low-dose treated samples is ineeping with the normal distribution of ferritin and hemosiderinn this normally hemocatheretic organ, the decreased reactiv-ty observed in high-dose treated samples and the concomitantresence of numerous megakaryocytes, suggest an increase ofemopoiesis. This fact, also evidenced after the treatment with-Naph-DNB (Invest New Drug, in press) probably reflects anmpairment of the bone marrow due to the treatment.

Similarly to 1-Naph-DNB, the isomer herein seems toroduce less consistent damages in the kidney, where somelterations of both tubules and vessels appeared at the highestoses.

In conclusion, our data demonstrate that the new molecule-Naph-DNB, an isomer originated by a modification of thetructure of the lead compound 1-Naph-DNB which essen-ially influences the molecular geometry, possesses appreciablentiproliferative, cytotoxic and pro-apoptotic activities togetherith a low profile of toxicity in vivo. Although our effort, as

xpected, did not solve the problem of the low solubility ofhe lead compound 1-Naph-DNB, they definitely demonstratehe possibility to increase the activity or modify the specificityf our nitrobutadiene compounds by simply modifying theireometrical array.

cknowledgments

This research was supported by funds from M.I.U.R. (FIRB001) and from the Universities of Genova and Bologna. C.

[

esearch 56 (2007) 318–328 327

revosto is recipient of a fellowship awarded by the Italianoundation for Cancer Research (FIRC).

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